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Identification of Escherichia Coli Genes Required For IDENTIFICATION OF ESCHERICHIA COLI GENES REQUIRED FOR BACTERIAL SURVIVAL AND MORPHOLOGICAL PLASTICITY IN URINARY TRACT INFECTIONS DANIEL GIUSEPPE MEDIATI A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy The ithree institute and School of Life Sciences, University of Technology Sydney November 2018 [blank page] Production Note: Signature removed prior to publication. iv TO MUM AND DAD, WHO INSTILLED IN ME THE GREAT VALUE OF EDUCATION. v CONTENTS IN BRIEF CHAPTER 1: INTRODUCTION CHAPTER 2: GENERAL MATERIALS AND METHODS CHAPTER 3: HIGH-THROUGHPUT SEQUENCING OF SORTED EXPRESSION LIBRARIES REVEALS INHIBITORS OF BACTERIAL CELL DIVISION CHAPTER 4: GENOME-WIDE COMPARISON OF E. COLI GENES REQUIRED FOR GROWTH IN COMPLEX AND MINIMAL MEDIA CHAPTER 5: THE ESSENTIAL GENE PROFILE SET OF UROPATHOGENIC E. COLI DURING BLADDER CELL INFECTION CHAPTER 6: DISCUSSION AND CONCLUSION REFERENCES vi TABLE OF CONTENTS Certificate of authorship and originality iii List of figures and supplementary data x List of tables and supplementary data xi Abbreviations xii Publications xiv Conference proceedings xv Acknowledgements xvi Preface xvii CHAPTER 1: INTRODUCTION 1 1.1 Urinary tract infections 3 1.1.1 Uropathogenic Escherichia coli 4 1.2 UPEC lifestyle from the intestine to the urinary tract 6 1.3 Infection cycle events in the bladder 8 1.3.1 Bacterial attachment and invasion of host cells 8 1.3.2 Intracellular bacterial communities 13 1.3.3 UPEC dispersal and filamentation 16 1.3.3.1 E. coli cell division and filamentation 19 1.3.3.2 Negative regulatory systems of cell division in E. coli 22 1.3.3.3 The SOS response and involvement of SulA 23 1.3.3.4 DamX and reversible filamentation during UTIs 26 1.4 UPEC metabolic responses during pathogenesis 27 1.4.1 The primary medium: nutritional aspects of urine 27 1.4.2 Central carbon metabolism 28 1.4.3 Amino acid catabolism 31 1.4.4 Iron uptake and transport systems 31 1.4.5 Other metabolic regulators of UPEC 33 1.5 Host immune responses to UPEC infection 34 1.5.1 UPEC evasion of host defences 34 1.6 ‘Omic screening approaches to further understand UTIs 35 1.6.1 Comparative bioinformatics 35 1.6.2 Transcriptomics 37 1.6.3 Functional genomics 38 1.7 Aims and objectives 40 CHAPTER 2: GENERAL MATERIALS AND METHODS 42 2.1 Bacterial strains and plasmids 43 vii 2.2 Bacterial and mammalian growth media 44 2.3 Chemicals, reagents and solutions 45 2.4 General growth conditions of E. coli 46 2.4.1 Preparation and transformation of E. coli 46 2.5 General mammalian growth conditions 47 2.5.1 General growth conditions of PD07i bladder cell infection model 47 2.6 General methods and protocols used in this thesis 48 2.6.1 Preparation and extraction of DNA from bacteria 48 2.6.2 Polymerase chain reaction (PCR) 48 2.6.3 Agarose gel electrophoresis 49 2.6.4 General multiplex DNA sequencing preparation 49 2.6.5 General analysis of DNA sequencing data 50 CHAPTER 3: HIGH-THROUGHPUT SEQUENCING OF SORTED EXPRESSION LIBRARIES REVEALS INHIBITORS OF BACTERIAL CELL DIVISION 51 3.1 Introduction 53 3.2 Results 55 3.2.1 A high-throughput screen based on flow cytometric sorting of DNA expression libraries 55 3.2.2 Identifying genomic regions that encode mediators of reversible bacterial filamentation 60 3.2.3 Identification and verification of DNA fragments that cause filamentation 67 3.3 Discussion 73 3.4 Methods 79 3.4.1 Bacterial strains and plasmids 79 3.4.2 Construction of the shotgun UTI89 genomic DNA expression library 79 3.4.3 Cell sorting for enrichment of filamentous clones from the expression library 80 3.4.4 High-throughput multiplex DNA sequencing and data analysis 80 3.4.5 Expression of cloned ORFs and analysis of cell phenotypes 82 3.4.6 Amplification and sub-cloning of fragments from identified genomic regions 82 CHAPTER 4: GENOME-WIDE COMPARISON OF E. COLI GENES REQUIRED FOR GROWTH IN COMPLEX AND MINIMAL MEDIA 84 4.1 Introduction 86 4.2 Results 88 4.2.1 Design of a modified TraDIS protocol 88 4.2.2 Construction of a transposon mutant library in UTI89 89 viii 4.2.3 Genes advantageous and disadvantageous for growth 92 4.2.4 Identification of genes required for growth in M9-glycerol minimal medium 95 4.2.5 Verification of genes required for growth in M9 102 4.2.6 Comparison of a subset of genes to E. coli K-12 104 4.3 Discussion 108 4.4 Methods 114 4.4.1 Bacterial strains and growth conditions 114 4.4.2 Construction of a mini-Tn5 transposon mutant library in UTI89 114 4.4.3 Mutant selection in minimal media 115 4.4.4 Transposon-directed insertion-site sequencing (TraDIS) 116 4.4.5 Analysis of nucleotide sequence data 116 4.4.6 Statistical analyses for identifying essential genes 117 4.4.7 Verification of individual deletion mutants 117 4.4.8 Transposon mutant library passage 118 4.4.9 Human bladder cell culture model of UTI 119 CHAPTER 5: THE ESSENTIAL GENE PROFILE SET OF UROPATHOGENIC E. COLI DURING BLADDER CELL INFECTION 120 5.1 Introduction 121 5.2 Results 123 5.2.1 Development of an up-scaled infection model to assay UPEC gene essentiality during UTIs 123 5.2.2 The essential gene profile set of UPEC during in vitro infection 130 5.2.2.1 The genes required for progression to IBCs 135 5.2.2.2 The genes required for UPEC dispersal and filamentation 141 5.2.2.3 The genes required for filament reversal and bacterial recovery from dispersal 151 5.3 Discussion 159 5.4 Methods 164 5.4.1 Bacterial strains and growth conditions 164 5.4.2 Development of an up-scaled in vitro UTI model 164 5.4.2.1 IBC phase of infection 164 5.4.2.2 Dispersal and filamentation phase of infection 165 5.4.3 Mutant harvest from infection for TraDIS analysis 166 5.4.3.1 Pre-infection (inoculate) sample 166 5.4.3.2 IBC sample 167 5.4.3.3 Dispersed bacterial sample 167 5.4.3.4 Non-dispersed bacterial sample 167 5.4.3.5 Recovery samples 168 ix 5.4.4 Transposon-directed insertion-site sequencing (TraDIS) 168 5.4.5 Analysis of nucleotide sequencing data 168 CHAPTER 6: DISCUSSION AND CONCLUSION 170 6.1 General Discussion 171 6.1.1 Reversible filamentation in UPEC 173 6.1.2 Metabolic adaptations and requirements of UPEC 175 6.1.3 Future directions 176 6.1.4 Concluding remarks 177 APPENDIX: SUPPLEMENTARY DATA 179 REFERENCES 188 x LIST OF FIGURES AND SUPPLEMENTARY DATA Figure 1.1 Well characterised virulence factors of uropathogenic E. coli. Figure 1.2 UPEC pathogenesis from the intestine to the urinary tract and bloodstream. Figure 1.3 Type-1 pilus and the adhesin protein FimH-mediated binding. Figure 1.4 Mouse bladder epithelial cell endocytosis of UPEC. Figure 1.5 Intracellular bacterial communities of UPEC within the mouse bladder. Figure 1.6 Bacterial filamentation, egress and dispersal from the bladder cell. Figure 1.7 Simplified overview of E. coli cell division and the main known negative regulators of Z-ring formation. Figure 1.8 FtsZ assembly during cell division and the involvement of SulA. Figure 1.9 Some important metabolic pathways of UPEC during urinary tract infection. Figure 3.1 Analysis and purification of filamentous bacteria from the UTI89 DNA- expression library by flow cytometry. Figure 3.2 High-throughput DNA sequencing of plasmid libraries from the reference- unsorted and filamentous-sorted populations. Figure 3.3 Overexpression of the pptE and pdhR ORFs cause E. coli filamentation independent of recA (SOS response). Figure 3.4 Partial fragments of ORFs cause filamentation. Figure 4.1 Genomic overview of the UTI89 transposon mutant library. Figure 4.2 Mutant compositional changes during library growth in LB. Figure 4.3 Number of essential genes according to COG functional category. Figure 4.4 Identification of genes required for growth of UPEC in M9 by TraDIS. Figure 4.5 Verification and characterisation of deletion mutants in UTI89. Figure 4.6 Characterisation of deletion mutants in E. coli K12 identified by TraDIS. Figure 5.1 Up-scaled bladder cell infection model permits UTI89 IBCs. Figure 5.2 Characterisation of UPEC dispersal in vitro and filamentous cells. Figure 5.3 Schematic diagram of TraDIS approach. Figure 5.4 The numbers of genes identified as important to stages of infection by TraDIS, indicating the numbers of genes common to multiple stages. Figure 5.5 Identification by TraDIS of UPEC genes required for bladder infection. Supp. Data Figure S1 Additional analyses of DNA sequencing data. Supp. Data Figure S2 pptE overexpression causes major effects on cell structure. Supp. Data Figure S3 ORFs that do not cause substantial filamentation when overexpressed. Supp. Data Figure S4 Outline of the modified TraDIS DNA sequencing protocol. Supp. Data Figure S5 Correlation between the Tni5 and Tni7 DNA sequencing ends. Supp. Data Figure S6 Analysis of filamentous bacteria from the dispersal phase of infection. xi LIST OF TABLES AND SUPPLEMENTARY DATA Table 2.1 General E. coli strains used in this thesis. Table 2.2 General plasmids used in this thesis. Table 2.3 Bacterial and mammalian growth media. Table 2.4 General chemical, reagents and solutions used in this thesis. Table 2.5 Aqueous buffers and solutions used in this thesis. Table 3.1 Read frequency from sequencing of plasmid libraries.
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